CN113288907A

CN113288907A - Application of iridoid compound in preparing anti-coronavirus medicine

Info

Publication number: CN113288907A
Application number: CN202110583442.3A
Authority: CN
Inventors: 甘礼社; 李冬利; 吴日辉; 金静维; 文燕; 李阳; 黄沛琦; 罗咏欣; 苏智成
Original assignee: Wuyi University
Current assignee: Wuyi University
Priority date: 2021-05-27
Filing date: 2021-05-27
Publication date: 2021-08-24
Anticipated expiration: 2041-05-27
Also published as: CN113288907B

Abstract

The invention discloses an application of iridoid compound in preparing anti-coronavirus medicine, which is characterized in that: the iridoid compound is cornus officinalis new glycoside, picroside III or new ligustrin. The iridoid compound of the scheme of the invention has good inhibitory action on coronavirus, has no obvious cytotoxicity, can be prepared into a medicament for clinically resisting the coronavirus, and has clear scientific value and practical significance for research and development of innovative medicaments for resisting the coronavirus, which have controllable quality, low toxicity and high efficiency.

Description

Application of iridoid compound in preparing anti-coronavirus medicine

Technical Field

The invention belongs to the technical field of medicinal chemistry, and particularly relates to an application of an iridoid compound in preparation of an anti-coronavirus medicament.

Background

Human Coronaviruses (HCoVs) are a class of RNA viruses with an envelope structure. The current HCoVs infecting humans are mainly 7 types, HCoV-229E, HCoV-NL63, HCoV-OC43, SARS-CoV, MERS-CoV, HCoV-HKU1 and SARS-CoV-2(Zhou P, Yang XL, Wang XG, et al. A pneumniaa outbreak associated with a new coronavirus of probabab origin. Nature 2020, Doi:10.1038/s 41586-020-. Among them, the outbreaks of Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and 2019 of a novel coronavirus (2019-nCoV or SARS-CoV-2) have been prevalent, which has brought a serious challenge to the social public health safety. Current clinical treatments for HCoVs infection are mainly symptomatic improvement. In terms of drug therapy, common antiviral drugs such as alpha-interferon, lopinavir, ribavirin and the like are used, and specific drugs directly aiming at HCoVs (a novel diagnosis and treatment scheme for coronavirus pneumonia (seventh edition of trial), national health committee, http:// www.nhc.gov.cn) are lacked. In contrast, the traditional Chinese medicine has a long history of practical application in epidemic prevention in China, and no matter in 2003, the traditional Chinese medicine has a long history of resisting SARS (CaoLi Juan, Wang, prevention and control of SARS by Chinese academy of sciences, Asia-Pacific medicine, 2014, 10(1), 1-3.) or the treatment of COVID-19 (Chengdui, Wen chrysanthemum, Liyi, Wudao winter, Zhongbiao, Song seven Yong, 51 novel patients with coronavirus pneumonia have been proved to have definite therapeutic effects and clinical significance in multi-center retrospective study, Tianjin medicine, 2020, http:// kns. cnki. net/kcms/detail/12.1349.R.20200310.1024.004.html), thereby greatly improving the death rate and having no serious toxic and side effects caused by the application of chemical drugs such as hormones.

Of the 7 HCoVs that are known to infect humans, HCoV-229E and HCoV-OC43 were found earlier, and were less toxic than HCoV-NL63, HCoV-HKU1, etc., which were later found, generally caused only acute upper respiratory infections, while having less effect on the lower respiratory tract, and infected patients often also showed only mild symptoms of colds (Gralinski LE, Baric RS. molecular Pathology of observing coronavirus infections. the Journal of Pathology 2015,235, 185 < 195 >). However, the other 3 kinds of HCoVs have strong toxicity, can cause acute infection symptoms of the lower respiratory tract, and have high mortality rate in part of severe patients.

The whole gene sequence alignment shows that the genomes of 7 human coronaviruses including SARS-CoV-2 have a great degree of similarity. The Coronavirus genome encodes the spinous process (spike, S) protein, the envelope (envelope) protein, the membrane (membrane) protein and the nucleocapsid (nucleocapsid) protein (Wilde AHD, snijet EJ, Kikkert M, et al. host factors in Coronavir reproduction in Tripp R., Tompkins S. (eds.) Roles of host gene and non-coding RNA expression in virus infection. Current Topics in Microbiology and Immunology, Springer, Cham 2018,419,1-42.), respectively. The latest research result shows that SARS-CoV-2 is mainly combined with host cell membrane receptor and membrane fusion through S protein mediated virus, and S protein is closely related to pathogenicity of virus; meanwhile, it has been identified that the functional protein receptor which mainly plays a role in the virus is angiotensin converting enzyme 2(ACE2) (Wan YS, Shang J, Graham R, et al. receptor reproduction by novel vary coronavir from Wuhan: An analysis based on restriction-growth students of SARS. journal of Virology,2020, DOI: 10.1128/JVIVI.00127-20.). SARS-CoV 3CL protease (3C-likeprotease, Mpro) is one of the major proteases of coronavirus, plays an important role in the replication process of virus, and can be used as a key target for drug design due to its high degree of conservation (Jo S, Kimm S, Shin DH, Kim MS. Inhibition of SARS-CoV 3CL protease by volatile acids. journal of Enzyme Inhibition and Medicinal Chemistry 2020,35(1), 145-151.). The roe and/Yanghai Tao subject group of the university of sea science and technology has successfully analyzed the crystal structure of the 3CL protease of SARS-CoV-2 (Liu X, Zhang B, Jin Z, Yang H, Rao, Z. the crystalline structure of COVID-19 main protease in complex with an in-hibitor N3.https:// www.rcsb.org/structure/6lu7), and has conducted virtual screening research (Maqing cloud, Liu, Du ocean, Zhang Gui Cheng, Liu Xuan, Liyun, Zi plum, Yang Yong, Sangrong, virtual screening of SARS-CoV-23 CL hydrolase Chinese medicine small molecule inhibitor and COVID-19 novel coronavirus pneumonia resistant Chinese medicine and compound prediction thereof based on high-flux molecular docking).

The traditional Chinese medicine considers that the disease position of the coronavirus pneumonia is mainly in the lung, spleen and stomach, the pathogenesis is the qi of epidemic crime, and the pathogenesis is mainly the disease caused by damp, cold, heat, toxin, stasis and deficiency. Xuluhua and the like (xuluhua, plum Rong, Zhengdan such as Shaobanfei, Wenxingqie, Linfengxia, Zengxiong, Song silver branch, researching the prescription medication rules of different stages of the novel coronavirus pneumonia based on 'due to time and circumstances', and Chinese experimental prescriptions journal. https:// doi.org/10.13422/j.cnki.syfjx.20201211) analyze the medication rules of traditional Chinese medicines used in the COVID-19 traditional Chinese medicine diagnosis and treatment scheme (including the national diagnosis and treatment scheme and the traditional Chinese medicine treatment scheme of various provinces), and find that the main medicines are tonifying medicines, heat clearing medicines, antidotes, dampness resolving medicines, phlegm-resolving medicines, cough-relieving medicines and asthma-relieving medicines and the like. Based on the analysis of modern action mechanism of traditional Chinese medicine, many herbs for tonifying deficiency, resolving dampness, resolving phlegm, relieving cough and asthma are related to immunity and relieving main symptoms, while heat-clearing herbs, including heat-clearing and toxicity-removing herbs, heat-clearing and fire-purging herbs, heat-clearing and dampness-drying herbs and heat-clearing and blood-cooling herbs, are related to antiviral action. Honeysuckle, rehmannia root, cape jasmine fruit, figwort root, large-leaved gentian, gentian and the like are heat-clearing traditional Chinese medicines used at high frequency in the COVID-19 traditional Chinese medicine diagnosis and treatment scheme. Research shows that Gentiana scabra can obviously improve the pathological effect caused by SARS-CoV infected Vero cells and can effectively inhibit SARS-CoV replication (Wen CC, Shuyur LF, Jan JT, et al. Traditional Chinese Medicine recipient extracts of Cibotium barometz, Gentiana scabra, Dioscorea batatas, Cassia tora, and Taxillus chinensis inhibition SARS-CoV replication. journal of Traditional and Complementary Medicine,2011,1(1), 41-50.); the gentiana macrophylla ethanol extract has a good effect of inhibiting the activity of SARS coronavirus main protease, and the iridoid compound obtained by further separation and purification can effectively inhibit SARS coronavirus main protease (von King Lei, Wang Bo Guo, Guo Yu, Chenwei Qiang, Yang Cheng and Fu Zi, research on effective components of gentiana macrophylla with SARS coronavirus main protease inhibitory activity, 28 th academic annual meeting of China chemical society, 2012 and 112.). A class of cycloolefine ether terpene alkaloids found in honeysuckle show good inhibitory effect on H3N2 influenza virus and Coxsackie virus (Yu Y, Zhu C, Wang S, Song W, Yang Y, Shi J. Homosoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoidoids with free of the flower bud of Longice japonica journal of Natural Products,2013,76, 2226-2233). Geniposide, a representative iridoid glycoside in gardenia, shows activity against H1NI influenza virus in both in vitro screening and in vivo tests, and can greatly reduce the levels of various inflammatory factors such as TNF-alpha and IL-6 (Zhang Y, Yao J, Qi X, Liu X, Lu X, Feng G. Geniposide antagonists anti-inflammatory and anti-viral activity against peptide A/Jiangsu/1/2009(H1N1) influenza virus infection in vitro and in vivo. antiviral Therapy,2017,22(7), 599-611.). The research shows that the traditional Chinese medicine for treating COVID-19 heat clearing contains one similar component: iridoid compounds have broad-spectrum antiviral activity, and can be used for treating SARS-CoV. Iridoid compounds are monoterpene compounds existing in various heat-clearing traditional Chinese medicines, and most of the compounds are glycoside components (Wangfenfei, Zhang 32895; Mei, Zhengxiao, Daizhe, Liu, Mashuangcheng, research progress on structure and biological activity of iridoid compounds, Chinese pharmacology, 2019, 33(3), 323-cake 330.). Research shows that iridoid compounds can protect the liver through CYP, Nrf2, ERK/MAPK and other channels; the compounds can play good roles of resisting inflammation and inhibiting the expression of inflammatory factors through NF-kB and other channels (the pharmacological action research of the compounds such as Zhang Hui Juan, Li Ju, Ma Xiao Hui, Li Tanman and secoiridoid glycosides is advanced, and the pharmacological research is 2018, 37(11), 659-channel 663).

In conclusion, the iridoid component is the material basis for the efficacy of various heat-clearing anti-coronavirus traditional Chinese medicines, and innovative anti-coronavirus medicines with controllable quality, low toxicity and high efficiency are expected to be found from the iridoid component, so that the novel anti-coronavirus medicine has clear scientific value and practical significance. However, the iridoid compounds are numerous, and not any iridoid compound can be applied to anti-coronavirus.

Statements in this background are not admitted to be prior art to the present disclosure.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a novel iridoid compound which can be used for resisting coronavirus.

According to one aspect of the invention, the application of the iridoid compound in preparing the anti-coronavirus medicines is provided, wherein the iridoid compound is cornuside, picroside III or neoligustrin. The 3 iridoid monomer compounds are respectively derived from fructus Corni mature fruit, rhizoma picrorhizae rhizome, and fructus Ligustri Lucidi plant.

In some embodiments of the invention, the coronavirus includes, but is not limited to, at least one of HCoV-229E, HCoV-NL63, HCoV-OC43, SARS-CoV, MERS-CoV, HCoV-HKU1, or SARS-CoV-2.

In some embodiments of the invention, the anti-coronavirus drug comprises a prophylactic or therapeutic coronavirus drug.

In some embodiments of the present invention, the raw materials for preparing the anti-coronavirus medicine further comprise pharmaceutical excipients. Can be prepared into different dosage forms by using common carriers.

In some embodiments of the invention, the pharmaceutical excipient is selected from at least one of a disintegrant, a diluent, a lubricant, a binder, a flavoring agent, a suspending agent, a surfactant, or a preservative.

In some embodiments of the invention, the disintegrant is selected from at least one of corn starch, potato starch, crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, carboxymethylcellulose calcium, or alginic acid; more preferably, the diluent is selected from at least one of lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate or crystalline cellulose; more preferably, the lubricant is selected from at least one of aerosil, magnesium stearate, calcium stearate, stearic acid, talc or anhydrous silica gel; more preferably, the binder is selected from at least one of acacia, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose or polyvinylpyrrolidone; more preferably, the flavoring agent is selected from at least one of aspartame, stevioside, sucrose, maltitol, or citric acid; more preferably, the suspending agent is selected from at least one of acacia, gelatin, methylcellulose, sodium carboxymethylcellulose, hydroxymethylcellulose, or aluminum stearate gel; more preferably, the surfactant is selected from at least one of lecithin, sorbitan monooleate, or glyceryl monostearate; more preferably, the preservative is selected from at least one of methyl paraben or propyl paraben.

In some embodiments of the invention, the pharmaceutical dosage form is in the form of a solid, semi-solid, or liquid; preferably an aqueous solution, non-aqueous solution, suspension or paste; more preferably tablets, capsules, soft capsules, granules, pills, oral liquids, emulsions, dry suspensions, dry extracts or injections.

According to another aspect of the invention, the application of the iridoid compound in preparing the coronavirus main protease inhibitor is provided, wherein the iridoid compound is cornuside, picroside III or neoligustrin.

According to another aspect of the invention, the application of the iridoid compound in preparing the 3CL protease inhibitor is provided, wherein the iridoid compound is cornuside, picroside III or neoligustrin.

According to a preferred embodiment of the present invention, at least the following advantages are provided: the iridoid compound of the scheme of the invention has good inhibitory action on coronavirus, has no obvious cytotoxicity, can be prepared into a medicament for clinically resisting the coronavirus, and has clear scientific value and practical significance for research and development of innovative medicaments for resisting the coronavirus, which have controllable quality, low toxicity and high efficiency. The three compounds of the scheme of the invention have good inhibitory activity on 3CL protease,wherein the inhibitory action of neoligustilide is strongest, and IC is₅₀14.340 μ M, it can inhibit 3CL protease activity, and has effect in blocking virus replication.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a graph showing the results of toxicity tests on compounds SDD-01 to SDD-08 in test examples of the present invention;

FIG. 2 is a graph showing the results of toxicity tests on compounds SDD-09 to SDD-16 in the test examples of the present invention;

FIG. 3 is a graph showing the results of toxicity tests for compounds SDD-17 to SDD-21 in the test examples of the present invention;

FIG. 4 is a graph showing the comparison of the inhibitory effects of the compounds SDD-01 to SDD-10 in the test examples of the present invention;

FIG. 5 is a graph showing the comparison of the inhibitory effects of the compounds SDD-11 to SDD-21 in the test examples of the present invention;

FIG. 6 is a graph showing the inhibition effect of cornuside and ligustilide, compounds of different concentrations, on SARS-CoV-2 in the test examples of the present invention.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

Example 1

Application of cornuside in preparing anti-coronavirus medicine is provided.

Example 2

Application of picroside III in preparing anti-coronavirus medicine is provided.

Example 3

Application of neoligustilide in preparing medicine for resisting coronavirus is provided.

Comparative example

Application of geniposide, oleuropein, catalpol, loganin, gentiopicrin, harpagoside, specnuezhenide, methyl deacetyl asperulate, digitoside D, paederosidic acid methyl ester, 8-O-acetyl shanzhiside methyl ester, picroside I, picroside, aucubin, monothioside, shanzhiside methyl ester, picroside II or harpagoside in preparing anti-coronavirus medicine.

The compounds in the above examples or comparative examples can be extracted and isolated from plants by the prior art, and the present examples are extracted by referring to the prior literature, and the specific reference information is shown in table 1 below (if the literature refers to a plurality of experimental conditions, the extraction is carried out by referring to the optimal experimental conditions).

TABLE 1

[1] Zhang Wen Juan, Limaoxing, Zhang quanlong, etc. research on rapid extraction and separation of geniposide and analgesic and anti-inflammatory effects [ J ] J.J. J.Experimental and formulary, 2012,18(21): 170-174).

[2] Queen jade, Swandena, Lihaiyan, etc. high speed countercurrent chromatography is used to separate oleuropein J from olive leaf, Chinese patent medicine 2018,40(04): 979-.

[3] Zhangbophoresis, Jiangzhong, Wangzhao, etc. UPLC/ESI-Q-TOF MS method analyzes chemical components [ J ] of fresh rehmannia root, dried rehmannia root and prepared rehmannia root, Chinese patent medicine 2016,38(05):1104 + 1108.

[4] Study on chemical components of iridoid glycosides in rehmannia glutinosa, including Ligeng, Wang Hui Sen, Liuming, etc. [ J ] Chinese medicine study, 2008(05):17-19.

[5]Deng S,West B J,Jensen C J.UPLC-TOF-MS Characterization and Identification of Bioactive Iridoids in Cornus mas Fruit[J].Journal of Analytical Methods in Chemistry,2013(8):710-972.)

[6]Wen CC,Shyur LF,Jan JT,et al.Traditional Chinese medicine herbal extracts of Cibotium barometz,Gentiana scabra,Dioscorea batatas,Cassia tora,and Taxillus chinensis inhibit SARS-CoV replication.Journal of Traditional and Complementary Medicine,2011,1(1),41-50.

[7] The separation and identification of chemical components in Tianjin Feng, Shangdong, Li Xuehang and Xuan Shen and the research on the hypoglycemic activity thereof [ J ] food industry science and technology, 2017,38(13):25-29.

[8] Songmeng break, Zhang Chuang lan, Li Huifen, etc. LC-TOF/MS identify chemical components [ J ] of simulated steaming product of 4 iridoid glycoside compounds in glossy privet fruit rapidly.

[9] Yongyong, Luoyangbo, research on chemical components of Hedyotis diffusa [ J ] Chinese herbal medicine, 2008(04):522 + 524.

[10] Xuxu, Lijian, Dinglisheng, et al research on chemical composition of Paederia scandens [ J ] J.Chinese J.2006, 31(17): 1436-.

[11] HPLC method is used for measuring iridoid glycoside and phenethyl alcohol glycoside [ J ] in lamiophlomis rotata root, Chinese herbal medicine 2011,42(02):279-281.

[12] Zhang Cheng Zhong, Li Chong, Shi Jian Gong, etc. iridoid glycoside [ J ] in Duyiwei of Tibetan medicine, Chinese herbal medicine, 1992,23(10): 509-.

[13]Hatano T,Yasuhara T,Abe R,et al.A galloylated monoterpene glucoside and a dimeric hydrolysable tannin from Cornus officinalis[J].Phytochemistry,1990,29(9):2975-2978.)

[14] Studies on chemical components of rhizome of picrorhiza scrophulariiflora Pennell, J. J.Chinese J., 2017,32(5):2070 and 2074.)

[15] Zhou Shi Liang, Zhang wen bin, Lie Luo xing, et al HPLC method determines the content of sesamoside in the Tibetan drug crab A [ J ]. Chinese pharmacy 2011,22(27): 2541-.

[16] Sun Chenzhi, Machilus thunbergii, Wangzasia, etc. separation and identification of chemical components of Forsythia suspensa [ J ] J. Med. Chemicals, 2021,31(04): 286-.

[17] Measuring the content of 10 ingredients before and after processing of morinda officinalis by UPLC-QQQ-MS method, wherein the Chinese medicinal materials comprise 2019,42(03) 588-.

[18]Choi J,Lee K T,Choi M Y,et al.Antinociceptive anti-inflammatory effect of Monotropein isolated from the root of Morinda officinalis.[J].Biological&Pharmaceutical Bulletin,2005,28(10):1915.

Test examples

This test example tested the natural iridoid compounds of the examples and comparative examples for their anti-coronavirus properties.

1. Cytotoxicity test

Weighing about 3g of natural iridoid compounds for cytotoxicity test, and dissolving the compounds in dimethyl sulfoxide (DMSO). The specific test process is as follows:

1) cell inoculation:

293T cells (commercially available) in logarithmic growth phase were adjusted to a cell density of 1 x 10^ 4/well, seeded at 100. mu.L/well in 96-well plates and cultured overnight in a 37 ℃ cell culture chamber.

2) Designing the concentration of the medicine:

4 concentration gradients were diluted 2-fold prior to dosing in DMEM medium containing 2% total volume of fetal bovine serum.

293T cells: the initial concentration was set at 100. mu.M (100, 50, 25, 12.5. mu.M), and 100. mu.L of the diluted drug per well was added to 293T cells in 96-well plates 1) at a final volume of 200. mu.L per well. 3 multiple wells were set for each drug concentration. The culture medium without the sample is used as a blank control, and oxymatrine is added as a positive control drug.

3) Detecting the absorbance:

after 48h incubation in the cell incubator, 20. mu.L of thiazolyl blue (MTT) (5mg/kg) working solution was added to each well and incubation was continued for 4 hours. The absorbance (OD) at 570nm was measured by a microplate reader.

4) And (3) survival rate calculation:

based on the measured OD values, the survival rate of 293T cells at each concentration of the drug was calculated, respectively, as compared with the control group. The results are shown in FIGS. 1 to 3.

The corresponding numbers, sample masses and solvent information in FIGS. 1-3 are shown in Table 2 below.

TABLE 2

This number is quoted throughout the source.

As can be seen from fig. 1 to 3, cytotoxicity of 21 monomeric compounds such as cornuside, picroside iii, neoligustilide and the like was evaluated using 293T cells as a model. The results show that the compounds have no obvious cytotoxicity in the concentration range of 12.5-100 mu M, the cell survival rate can reach more than 75%, the toxicity is low, and the safety is high.

2. anti-HCoV-OC 43 infection activity of natural iridoid

1) HRT-18 cells were seeded at a density of 4 x 10^5 cells per well in 12-well plates at 37 ℃ with 5% CO₂The culture was carried out overnight.

2) Removing supernatant, washing cells twice with PBS buffer, mixing 500 μ l of corresponding concentration drug with 500 μ l of HCoV-OC43 diluted to 0.2 concentration MOI, standing at 33 deg.C and 5% CO₂And (5) incubating for 24h in a cell incubator.

3) The supernatant was removed and the cells were washed twice with PBS buffer. Adding the corresponding concentration of the drug solution, and continuing to add 5% CO at 33 deg.C₂Incubation in cell incubator for 72 h.

4) RNA was collected and extracted, as referenced to Cell Total RNA isolation Kit (Takara).

5) Reverse transcription of RNA into cDNA, reverse transcriptase: takara Prime script Mreagent Kit (Cat: RR 036A).

6) And (5) carrying out qRT-PCR detection to detect the change of the relative expression quantity of the virus NP mRNA.

7) The reaction was prepared on ice as follows in table 3:

TABLE 3

The primer sequences are as follows:

OC43 NP protein upstream Primer (Forward Primer, shown in Seq No. 1): CGGTCTGGTAATGGCATCCTCAAG

OC43 NP protein downstream Primer (Reverse Primer, shown in Seq No. 2): CATTCCCTCCTGATGGTTGCTGAG

And (3) computer detection: ABI 7500 quantitative PCR instrument;

pre-denaturation: 95 ℃, 30 seconds, 1 cycle;

and (3) PCR amplification: at 95 ℃, 15 seconds, 40 cycles;

annealing: 60 ℃ for 1 min; and (6) recording.

The histogram of the inhibition ratios of the different compounds is shown in FIGS. 4-5. As can be seen from the figure, the medicine screening of in vitro anti-coronavirus HCoV-OC43 is carried out on iridoid compounds within a safe concentration range (12.5-100 mu M), and oxymatrine (CQ) is used as a positive control, the result shows that the inhibition rate of compounds cornuside and picrorhiza rhizome III on HCoV-OC43 can reach more than 50% at 20 mu M, neoligustilide also has certain inhibition activity at 20 mu M, and other compounds have no inhibition effect on HCoV-OC43 except part of the compounds with partial activity at 20 mu M, so that only the neoligustilide, the cornuside and the picrorhiza rhizome III have stable inhibition effect on HCoV-OC 43.

3. Activity test against SARS-CoV-2 pseudovirus infection

The S protein of SARS-CoV-2 is transfected by HEK-293T cell to form SARS-CoV-2 pseudovirus, and the inhibitory effect of iridoid compounds on SARS-CoV-2 pseudovirus is determined. The specific test process is as follows:

1) SARS-CoV-2S protein (pNL4-3.Luc. R-E-pcDNA3.1-SARS-CoV-2-Sipke) virus packaging:

HEK-293T cells in logarithmic growth phase 4 x 10^ 5/ml, 2ml per well are uniformSeeded in 6-well plates. 37 ℃ and 5% CO₂The cells were cultured in a cell incubator for 24 hours. The fresh medium was changed 1 hour before transfection, and 100. mu.l of blank DMEM medium was used to prepare plasmid diluent and transfection reagent (Polyjet) diluent, in the following proportions (plasmid DNA was extracted using a endotoxin-removing extraction kit (commercially available)):

pNL4-3.Luc. R-E-vector plasmid 1000ng

pcDNA3.1-SARS-CoV-2-Sipke 500ng

PolyJet 6μl

The preparation method comprises the following steps: the pNL4-3.Luc. R-E-plasmid and pcDNA3.1-SARS-CoV-2-Sipke plasmid were added into 100. mu.l of blank DMEM medium at the same time and mixed, and Polyjet was diluted with 100. mu.l of blank DMEM medium and mixed. Adding the PolyJet diluent into the plasmid diluent, uniformly mixing, incubating for 15 minutes at room temperature, uniformly adding into HEK-293T cells, culturing for 48 hours at 37 ℃, collecting supernatant virus liquid, centrifuging for 10 minutes at 4000rpm, and filtering by using a 0.45-micrometer sterile filter head to obtain the SARS-CoV-2 pseudovirus.

2) Pseudovirus inhibition experiments: and selecting safe concentration of the compound according to the cytotoxicity experiment result for activity detection.

Drug and pseudovirus effects: 293T cells (293T/ACE2) stably overexpressing human SARS-CoV-2 receptor ACE2 in logarithmic growth phase were plated evenly in 96-well plates at 1 × 10^ 4/well. Cultured in a cell culture chamber at 37 ℃ for 24 hours.

The initial concentration of the sample was set at 20 μ M and 3 concentration gradients (20, 10, 2 μ M) were diluted with DMEM medium containing 2% total fetal bovine serum volume before administration, each concentration being 3 replicate wells. Adding pseudovirus diluent into the medicine after gradient dilution, uniformly mixing, acting for 30 minutes at room temperature, adding 100 mu l/hole into ACE2/293T cells, and continuously culturing for 48 hours at 37 ℃.

And (3) detection: the medium was removed, and the cells were washed once with 200. mu.l/well of sterile PBS (pH7.4), 40. mu.l of 1 × Cell lysate (1 × Cell lysis) was added to each well, and lysed by shaking at room temperature for 15 minutes. Transfer 30. mu.l/well of the lysed supernatant to a 96-well white microplate and detect according to single luciferaseAdding an isovolumetric diluted Luciferase substrate into the instruction book of the kit (Luciferase Assay System), immediately carrying out value detection luminescence by an enzyme label instrument, and judging the activity of the sample for inhibiting virus adsorption according to the luminescence value. Calculating Inhibition rate (Inhibition rate) according to the corresponding relation between the luminous value and the drug concentration, drawing a curve and calculating the half Inhibition concentration IC of the iridoid compound₅₀. The results of the experiment are shown in FIG. 6.

As can be seen from FIG. 6, 21 monomeric compounds such as cornuside, picroside III, and neoligustilide are tested for anti-SARS-CoV-2 activity in vitro in a safe concentration range, and in a pseudovirus entry experiment, oxymatrine (CQ), which is a known drug capable of inhibiting SARS-CoV-2PsV from entering cells, is used as a positive control, and the results show that the cornuside, the picroside III, and the neoligustilide have certain effects of inhibiting SARS-CoV-2PsV from infecting 293T/ACE2 cells, and the inhibition rate is higher than that of other compounds, and can reach more than 50%. In the in vitro drug screening of anti-coronavirus HCoV-OC43, known oxymatrine (CQ) is used as a positive control, and the result shows that the inhibition rate of cornuside and picroside III on HCoV-OC43 can reach more than 50% at 20 mu M. Comparison of Positive control drug oxymatrine (IC)₅₀20.985 μ M), Corni fructus neoside (SDD-12) and neoligustilide (SDD-15) have effects of inhibiting SARS-CoV-2PsV infection of 293T/ACE2 cell, with inhibition rate of over 50%, and its IC is presumed₅₀Less than 20. mu.M.

4. Effect test on SARS-CoV-2 Virus 3CL protease Activity

1) The experimental principle is as follows:

fluorescence Resonance Energy Transfer (FRET), the activity of the 3CL protease is detected by measuring fluorescence. One donor group (EDANS) and a quencher group (DABCYL) were both attached to the natural substrate of the 3CL protease, and DABCYL quenches EDANS when the substrate is not cleaved, so that no fluorescence is detected. When this substrate was cleaved by 3CL protease, EDANS was no longer quenched by DABCYL, and EDANS fluorescence was immediately detectable.

2) Preparation of reagents, consumables and instruments:

3CL proteinase (Nickel column purification)

3C substrate (GL Biochem, Cat No:: 730985-86-1)

Tris(Sangon Biotech，Cat No:A600194)

EDTA(Sangon Biotech，Cat No:B540625)

DMSO(TCI，Cat No:D5293)

Positive control oxymatrine (MCE, HY-13750, DMSO was used for the same test)

384-well Solid White Flat Bottom(Corning，Cat No:3701)

Enzyme-labeling instrument (company: Molecular Devices, model: SpectraMax Paradigm)

Centrifuge (company: Beckman, model: Allegra-15R)

Oscillator (company: Scilogex, model: XW-80A)

3) Reaction System (20. mu.l)

The drug concentrations and basic configuration information are shown in table 4 below:

TABLE 4

4) Reaction process

4.1200 × preparation of compound solution:

transverse gradient dilution: compound stock (20mM) was diluted 3-fold in DMSO in 9 concentration gradients (3. mu.l compound + 6. mu.l DMSO) at 20mM, 6.667mM, 2.222mM, 0.741mM, 0.247mM, 0.082mM, 0.027mM, 0.009mM, 0.003mM, respectively.

4.2 the 200 × compounds with 9 concentration gradients are respectively diluted by 20 times to 10 × compounds (2 μ l compound +38 μ l Buffer) with Buffer longitudinally, then 2 μ l of 10 × compounds are respectively added into the final reaction system (20 μ l), and diluted by 10 times to 1 × compounds, that is, the final concentrations of the compounds are the concentrations to be measured: 100 μ M, 33.333 μ M, 11.111 μ M, 3.704 μ M, 1.235 μ M, 0.412 μ M, 0.137 μ M, 0.046 μ M, 0.015 μ M;

experimental groups: 3CL proteinase (1. mu.l) + Drug (2. mu.l) + buffer (15. mu.l), reacted at room temperature for 30 min;

blank control (0%): buffer (18. mu.l), room temperature reaction for 30 min;

DMSO control (100%): 3C (1. mu.l) + DMSO (2. mu.l) + buffer (15. mu.l), and reacted at room temperature for 30 min.

4.3 Add 2. mu.l of 3C substrate per well, after a short centrifugation in 384 well plates, react for 20min at room temperature in the dark.

4.4384 well plate is put into enzyme labeling instrument, Tune cartridge is selected, FRET mode is adopted, reading parameters are set:

λ_exc/λ_em＝360nm/490nm。

4.5 operating the microplate reader, continuously reading for 60min, and collecting data every 10 min.

4.6 at the end of the reading, the data at which the enzyme activity reached the highest value were selected as the final treatment data (RLU).

5) Data processing formula

The inhibition rate of the test compound was calculated by the following formula:

Inhibition(％)＝100％-(RLU_compound-RLU_0％control)/(RLU_dmso _control-RLU _0％control)*100％

inhibition was calculated for different concentrations of compound in Excel, and GraphPad Prism software was used to plot inhibition and IC₅₀The results are shown in Table 5 below.

TABLE 5

As can be seen from Table 4, compared with the positive control oxymatrine, the natural iridoid compounds of the embodiment of the invention have obviously better effects than oxymatrine, especially the inhibitory effect of ligustrazine on 3CL protease is strongest, and IC is₅₀Can reach 14.340 mu M, and can inhibit 3CLThe protease activity, thereby realizing the effect of blocking virus replication and having good application prospect.

The experimental process utilizes the current pharmaceutical chemistry and molecular biology popular technologies, takes the active ingredients of a class of traditional Chinese medicines as research objects and molecular probes, develops drug discovery on human coronavirus diseases for research, and lays a material foundation for discovering anti-coronavirus innovative drugs with controllable quality, low toxicity and high efficiency. From the innovation of the research idea, the invention takes the analysis of the prescription of the traditional Chinese medicine for clinically treating SARS/COVID-19 as the starting point, and provides the systematic research on the active ingredients of the traditional Chinese medicine by deeply thinking the traditional Chinese medicine so as to discover the innovative medicine. The theory of the traditional Chinese medicine idea and the modern pharmaceutical science are mutually collided, and the innovation assumption is made on the basis of the previous research and research results, so that various heat-clearing traditional Chinese medicines are provided to play the effect of resisting coronavirus through iridoid components.

In conclusion, the neoligustrazine inhibition, the cornuside and the picroside III have different inhibition effects on different coronin and have certain activity on 3CL protease, so the compound has good application prospect in resisting coronavirus. The comparison between the above test process and other natural iridoid compounds with potential anti-coronavirus activity, which are separated in the related art, proves that the iridoid compound provided by the scheme of the embodiment of the invention has better medicinal activity. The compounds are represented by SARS-CoV-2 and HCoV-OC43 in human coronavirus, and pharmacological activity research shows that the compounds have certain effect of inhibiting SARS-CoV-2PsV from infecting 293T/ACE2 cells; in the in vitro screening of the medicine for resisting coronavirus HCoV-OC43, the two monomeric compounds of cornuside and neoligustilide have stronger inhibition effect on HCoV-OC 43. The invention takes SARS-CoV-2 virus 3C main protease as target to carry out activity analysis, the activity of the ligustilide for inhibiting 3C protease is stronger than that of cornuside and picroside III, and IC₅₀14.340 μ M, it can inhibit 3C protease activity, and has effect in blocking virus replication.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

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Application of iridoid compound in preparing anti-coronavirus medicine

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Claims

1. The application of the iridoid compound in preparing the anti-coronavirus medicine is characterized in that: the iridoid compound is cornus officinalis new glycoside, picroside III or new ligustrin.

2. Use according to claim 1, characterized in that: the coronavirus includes, but is not limited to, at least one of HCoV-229E, HCoV-NL63, HCoV-OC43, SARS-CoV, MERS-CoV, HCoV-HKU1, or SARS-CoV-2.

3. Use according to claim 1, characterized in that: the anti-coronavirus drug comprises a preventive or therapeutic coronavirus drug.

4. Use according to claim 1, characterized in that: the raw materials for preparing the anti-coronavirus medicine also comprise pharmaceutic adjuvants.

5. Use according to claim 4, characterized in that: the pharmaceutic adjuvant is selected from at least one of a disintegrating agent, a diluting agent, a lubricating agent, an adhesive, a flavoring agent, a suspending agent, a surfactant or a preservative.

6. Use according to claim 5, characterized in that: the disintegrating agent is selected from at least one of corn starch, potato starch, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, calcium carboxymethyl cellulose or alginic acid; more preferably, the diluent is selected from at least one of lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate or crystalline cellulose; more preferably, the lubricant is selected from at least one of aerosil, magnesium stearate, calcium stearate, stearic acid, talc or anhydrous silica gel; more preferably, the binder is selected from at least one of acacia, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose or polyvinylpyrrolidone; more preferably, the flavoring agent is selected from at least one of aspartame, stevioside, sucrose, maltitol, or citric acid; more preferably, the suspending agent is selected from at least one of acacia, gelatin, methylcellulose, sodium carboxymethylcellulose, hydroxymethylcellulose, or aluminum stearate gel; more preferably, the surfactant is selected from at least one of lecithin, sorbitan monooleate, or glyceryl monostearate; more preferably, the preservative is selected from at least one of methyl paraben or propyl paraben.

7. Use according to claim 1, characterized in that: the pharmaceutical dosage form is in solid, semi-solid or liquid form; preferably an aqueous solution, non-aqueous solution, suspension or paste.

8. Use according to claim 7, characterized in that: the medicament dosage form is tablets, capsules, soft capsules, granules, pills, oral liquid, emulsion, dry suspension, dry extract or injection.

9. The application of iridoid compound in preparing coronavirus main protease inhibitor is characterized in that: the iridoid compound is cornus officinalis new glycoside, picroside III or new ligustrin.

10. The application of the iridoid compound in preparing the 3CL protease inhibitor is characterized in that: the iridoid compound is cornus officinalis new glycoside, picroside III or new ligustrin.